Influence of isoproterenol on net potassium uptake in whole pigeon erythrocytes in vitro

Isoproterenol increases net uptake of potassium in whole pigeon erythrocytes $\text{in vitro}$; effect of 10^?5 M isoproterenol is blocked by 10^?4 M propranolol. Pentifylline, a potent inhibitor of cAMP-phosphodiesterase, significantly amplifies effect of isoproterenol, indicating that isoproterenol-effect is mediated by cAMP. cAMP alone has no direct influence on net potassium uptake, while dibuturyl-cAMP has a very weak effect. Isoproterenol-effects are also mediated by the cell membrane protein-phosphorylation.     

Rat hepatoma 5123TC albumin mRNA directs the synthesis of pre-proalbumin identical to rat liver pre-proalbumin.

Effect of hemin, mild periodate oxidation and concanavalin A (Con A) on $\text{in vitro}$ biosynthesis of membrane proteins and hemoglobin, in the rabbit reticulocyte, was examined. Whereas addition of hemin to the incubation medium stimulates synthesis of both hemoglobin and membrane proteins, addition of Con A, at concentrations which agglutinate cells, selectively stimulates membrane protein biosynthesis. Mild periodate treatment of cells inhibits synthesis of hemoglobin and membrane proteins; this inhibition is not related to oxidation of a membrane component since hemoglobin synthesis in a cell free lysate of treated cells is similarily inhibited.     

On the possibility of carnitine binding within the brown adipose tissue cell

Even though injected radioactive carnitine is found to accumulate in brown adipose tissue of suckling rats, no consistent specific binding to a protein in the high speed supernatant of this tissue could be demonstrated, either $\text{in vivo}$ or $\text{in vitro}$. On $\text{in vitro}$ incubation of $\text{in vivo}$ prelabelled brown fat, 80% of the label was released into the medium within 20 minutes.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

Uptake of ribosomal proteins by isolate HeLa nuclei.

The uptake of radioactive ribosomal proteins by isolated HeLa cell nuclei has been studied. Ribosomal proteins are taken up by nuclei $\text{in vitro}$ more rapidly than are cytosol proteins, suggesting that the uptake is selective. In addition, the ribosomal proteins are found associated with the nucleolus to a greater extent than are the cytosol proteins.     

Transferrin acquisition of catabolized erythrocyte iron in the rat

Rat plasma contains two isotransferrins rather than the single iron-binding protein found in plasma of other species, and it was recently proposd that differences between the biological behavior of each isotransferrin accounted for observations previously attributed to behavioral differences between each of the two transferrin iron-binding sites. The two isotransferrins were isolated from rat plasma by DEAE-Sephadex ion-exchange chromatography and isoelectric focusing. The pH-dependent iron-dissociating and reticulocyte iron-donating properties of each isotransferrin were investigated and found to be indistinguishable. Like human transferrin, one iron-binding site retains its affinity for iron below pH 6 and this property was used to investigate the $\text{in}$$\text{vivo}$ acquisition of catabolic iron in order to determine whether the process occurs at one specific or both binding sites. Plasma radioactive iron, derived from injected ⁵⁹Fe-labelled heat denatured erythrocytes was bound with high specificity to the transferrin iron-binding site that was most resistant to acidic dissociation. This finding supports Fletcher and Huehns' hypothesis that each of the two rat transferrin iron-binding sites is endowed with a separate functional role.     

Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase

The mechanism of transferrin uptake by reticulocytes was investigated using rabbit transferrin labelled with ¹²⁵I and ⁵⁹Fe and rabbit reticulocytes which had been treated with trypsin, Pronase or neuraminidase. Low concentrations of the proteolytic enzymes produced a small increase in transferrin and iron uptake by the cells. However, higher concentrations or incubation of the cells with the enzymes for longer periods caused a marked fall in transferrin and iron uptake. This fall was associated with a reduction in the proportion of cellular transferrin which was bound to a cell membrane component solubilized with the non-ionic detergent, Teric 12A9. The effect of trypsin and Pronase on transferrin release from the cells was investigated in the absence and in the presence of $\text{N-}\text{ethylmaleimide}$ which inhibits the normal process of transferrin release. It was found that only a small proportion of transferrin which had been taken up by reticulocytes at 37°C but nearly all that taken up 4°C was released when the cells were subsequently incubated with trypsin plus $\text{N-}\text{ethylmaleimide}$, despite the fact that about 80% of the ⁵⁹Fe in the cells was released in both instances. Neuraminidase produced no change in transferrin and iron uptake by the cells.These experiments provide evidence that transferrin uptake by reticulocytes requires interaction with a receptor which is protein in nature and that following uptake at 37°C, most of the transferrin is located at a site unavailable to the action of proteolytic enzymes. The results support the hypothesis that transferrin enters reticulocytes by endocytosis.     

Increased production of the outer membrane receptors for colicins B, D and M by Escherichia coli under iron starvation.

Growth of $\text{E. coli}$ K-12 under severe iron stress results in increased production of the outer membrane receptors for colicins B, D, Ib and M. The increase in colicin receptor activity coincides with the appearance of large amounts of two high molecular weight proteins in the outer membrane of the cells. These proteins are identified as the outer membrane receptors for colicins B and D and for colicin M. Mutants lacking a functional outer membrane receptor for colicins B and D are defective in the uptake of iron complexed with the siderochrome enterochelin, and are thus comparable with $\text{tonA}$ mutants which lack a functional receptor for colicin M and are defective in the uptake of iron complexed with ferrichrome (6). The colicin B and D receptor may therefore function in the uptake of ferri-enterochelin.     

Differences in uncoupling effects associated with the uptake of lactose and dansyl-galactoside in Escherichia coli membrane: Active transport versus specific binding

Cytoplasmic membrane vesicles isolated from Escherichia coli take up dansyl-galactoside, a fluorescent competitive inhibitor of lactose transport, to much lower levels than lactose. An initial interpretation, based on the study of the fluorescent changes accompanying the energy-dependent uptake, was that it represented a one-to-one specific binding to the lac carrier protein which was not followed by transport. Recently, on the basis of a new estimation of the number of lac carrier in the membrane, it has been advanced that the uptake of dansyl-galactoside represents a nonspecific binding on the inner surface of the membrane following transport. We discriminate between the two interpretations by comparing the effects of lactose and dansyl-galactoside uptake on the electrochemical gradient of protons ($\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$), generated by the oxidation of substrates, and on the uptake of proline. Indeed, it is known that the rate of lactose transport is such that it leads, as a consequence of the lactose/H⁺ symport, to an observable decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$, and secondary to this decrease to an inhibition of the uptake of proline transported at much lower rate. We show that the rates of uptake of lactose and dansyl-galactoside by the membrane vesicles are similar; yet the uptake of dansyl-galactoside does not lead to the uncoupling effects which are associated with the uptake of lactose. We discuss the possible reasons for the absence of this uncoupling effect, and we conclude that our data are incompatible with the notion that the energy-dependent uptake of dansyl-galactoside is associated with an active transport involving a dansyl-galactoside/H⁺ symport. On the contrary, the data substantiate the initial interpretation that the energy-dependent uptake of dansyl-galactoside reflects the binding to the lac carrier not followed by transport.     

Suppression of iron uptake deficiency in Escherichia coli K-12 by loss of two major outer membrane proteins.

A novel iron uptake system was observed in pseudorevertants of $\text{Escherichia}$,$\text{coli}$ strains defective in ferrienterochelin transport. The new system is unique in that it is an active transport system that does not utilize any known siderophore. Acquisition of the new uptake system occurs concomitantly with the loss of two major outer membrane proteins (b and c) believed to function as structural components of transmembrane pores.     

Binding of hexachlorobenzene to erythrocytes: Species variation

An $\text{in vitro}$ study revealed that hexachlorobenzene binds to the erythrocytes of rats, mice and rabbits, but that there is little or no such binding in 14 other species, including humans and rhesus monkeys. This binding is not a general phenomenon for all chlorinated hydrocarbon pesticides, since similar experiments using ¹⁴C-DDT and ¹⁴C-Mirex have failed to demonstrate this effect. The structural integrity of the erythrocytes may be important in this binding process since hexachlorobenzene binds poorly to rat ghost cell membranes.     

Glucocorticoid tight binding in rat liver and thymus particles

Glucocorticoid binding to certain cell particles of rat liver and thymus following treatments $\text{in vivo}$ and $\text{in vitro}$ consists in part of a very “tight binding” that resists hot and cold perchloric acid extractions. This binding is found in thymus nuclei and in liver cytoplasmic particles, but not in liver nuclei nor in thymus mitochondria or microsomes. The existence of “tight binding” coincides with the ability of the same particles to bind free corticoid directly in incubations $\text{in vitro}$. The difference in the cellular location of this binding suggests that different methods of glucocorticoid activation exist in the anabolic target tissue, liver, and the catabolic target tissue, rat thymus.     

The abnormal phosphorylation of spectrin in human hereditary spherocytosis

The phosphorylation of the proteins of the erythrocyte membrane of patients suffering from hereditary spherocytosis is investigated in intact erythrocytes by their incubation in the presence of radioactive inorganic phosphate. Examination of the phosphorylated components by high-resolution two-dimensional gel electrophoresis reveals only one defect in the pathological membranes, a depressed phosphorylation of the smaller polypeptide of spectrin; band 2. The phosphorylation of band 2 is measured with reference to the phosphorylation of syndein ($\text{2.1 + 2.2 + 2.3}$). In patients showing overt clinical symptoms and for whom splenectomy is advocated the phosphorylation of band 2 is depressed by approx. 70%. After splenectomy the phosphorylation of membrane proteins is restored to normal levels.     

Glycosaminoglycans are integral constituents of renal glomerular basement membrane

Basement membranes from canine renal glomeruli were isolated following osmotic lysis and sequential detergent treatment. Substantial amounts of uronic acid in unfractionated membranes were demonstrated with the carbazole and orcinol reactions. About 10–15% of basement membrane uronic acid was solubilized with neutral salt solutions. Denaturation in 8M urea solubilized ?70% of the uronic acid but only ?10% of basement membrane hydroxyproline; the latter was solubilized after reduction and alkylation. Uronic acid containing glycoprotein isolated by denaturation did not bind to carboxymethylcellulose and migrated as a high molecular weight band on SDS-gel electrophoresis. The ability of isolated rat glomeruli to incorporate radioactive sulfate $\text{in}\text{vitro}$ was demonstrated. These findings indicate that sulfated glycosaminoglycans are integral components of glomerular basement membrane.     

Increased acridine orange uptake and lysosomal enzyme levels in rat liver after steroid administration

A steroid which stabilizes lysosomes $\text{in vitro}$ and a pyrogenic steroid which labilizes lysosomes $\text{in vitro}$ were compared with respect to their ability to modify lysosomal uptake and lysosomal enzyme levels $\text{in vivo}$. Cortisone acetate increased the uptake of acridine orange by rat liver lysosomes when the dye was administered by intrathoracic injection. The steroid increased and accelerated the uptake of acridine orange so that, in liver lysosomes from treated rats, the maximum uptake was double that of controls and was reached at 2h, whereas in controls the maximum uptake was at 4h after the injection of the dye. This large elevation of uptake is specific to the lysosomal fraction and is not seen in other subcellular fractions of rat liver. The specific activities of a lysosomal enzyme β-N-acetylglucosaminidase were increased in lysosomal fractions from cortisone acetate-treated rats. Etiocholanolone, a steroid which labilizes lysosome $\text{in vitro}$, similarly accelerated and increased acridine orange uptake by lysosomes but had little effect on lysosomal β-N-acetylglucosaminidase levels. Thus the ability of steroids to stabilize or labilize lysosomes $\text{in vitro}$ does not correlate with their effect on lysosomal uptake of injected substances $\text{in vivo}$, or with their ability to induce increased specific activities of lysosomal enzymes.     

t-Butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes

Treatment of human erythrocytes with micromolar concentrations of $\text{t-}\text{butyl}$ hydroperoxide causes a variety of changes in the physical properties of the cells. Red cells exposed to concentrations of $\text{t-}\text{butyl}$ hydroperoxide of less than 750 μM for 15 min exhibited significant decreases in cellular and membrane deformability, increases in membrane-associated protein crosslinking, osmotic fragility and the viscosity of the intracellular hemoglobin solution. No changes in the volume or density of the cells were observed. Changes in cellular deformability are probably attributable solely to changes in the mechanical properties of the cell membrane. Conversely, when red cells are exposed to $\text{t-}\text{butyl}$ hydroperoxide concentrations in excess of 750 μM for 15 min they exhibited decreases in cellular deformability which may be related to increases in cell volume as well as membrane rigidity.     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

Interactions of lead and calcium on the synaptosomal uptake of dopamine and choline

Exposure of rodents to lead $\text{in vivo}$ has been associated with alterations in cholinergic and dopaminergic neurotransmission in the CNS. These effects have been hypothesized to result from competitive interactions between lead and calcium at sites involved in uptake and release of neurotransmitters and their precursors. These experiments reproduced the $\text{in vivo}$ observation by $\text{in vitro}$ exposure of crude synaptosomal suspensions to lead. Lead-induced inhibition of high affinity choline uptake was mimicked by reduced in vitro calcium concentrations, which suggests that lead's effects on cholinergic function are explainable by the lead-calcium hypothesis. However, inhibition of dopamine uptake was produced only by lead and not by reduced calcium; further additions of calcium did not reverse lead-induced effects on dopamine uptake. Increased calcium concentrations were shown to increase the release of dopamine; lead in the presence of normal calcium concentration did not affect dopamine release. However, more dopamine was released when increased calcium was combined with exposure to 1 × 10^?4 lead. This effect may have resulted from lead's ability to increase the uptake of calcium by synaptosomes. Thus, the interactions between lead and calcium appear to differ in terms of effects on cholinergic and dopaminergic function; in the former, the results suggest a competitive interaction similar to that shown functionally at peripheral cholinergic sites; in the latter, a different role for calcium is hypothesized which may account for the different effects of lead.     

Isolation and amino acid composition of two somatostatin-like peptides from ovine hypothalamus: somatostatin-28 and somatostatin-25

Two $\text{b}$-type cytochrome components, differing in their mid-point redox potentials as well as their reactivities toward CO, are demonstrated in the membrane particle of $\text{Azotobacter}\text{vinelandii}$ by potentiometric titration. The low redox component is revealed to be CO-reactive while the higher redox component is not. Accordingly, the later cytochrome is believed not to be directly involved in the reduction of oxygen while the former component is the cytochrome $\text{o}$ of $\text{Azotobacter}$.     

Selenium binding proteins in rat tissues

The 100,000 × g extracts of rat intestine and colon were incubated $\text{in}\text{vitro}$ with Na₂[⁷⁵Se]O₃. Chromatography of this material on a Sephadex G-100 column produced three radioactive peaks corresponding to molecular weights of 17,000, 68,000 and > 90,000. The 17,000 peak corresponded to a protein which sedimented in the 2S region of a 5–20% ($\text{w}\text{v}$) linear sucrose density gradient. Selenium binding to this protein was specific, stable and sensitive to thiol inhibitors such as p-chloromercuriphenylsulfonic acid (1 mM) and iodoacetamide (2 mM). Chromatography of rat serum - [⁷⁵Se] complex on Sephadex G-100 yielded only two radioactive peaks that corresponded to molecular weights of 68,000 and > 90,000. The 2S selenium binding protein of intestine and colon may mediate the biological functions of selenium in those tissues.